In the past year, we have advanced the objectives of this project along several lines. [unreadable] [unreadable] (1) Development of a reverse genetics system. Two years ago, the Taniguchi laboratory described a helper virus-dependent, single-gene replacement reverse genetics system for rotavirus (Komoto et al, 2006). This system used a replication-deficient vaccinia virus (rDIs-T7pol) as the source of T7 RNA polymerase (T7pol) to drive the transcription of a SA11 rotavirus VP4 gene from a transfected cDNA plasmid. Infection of the transfected cells with a helper rotavirus (KU strain) allowed for the recovery of recombinant viruses that had incorporated the SA11 VP4 gene into the KU genome. [unreadable] [unreadable] Shortly thereafter, the Dermody laboratory reported the development of a fully plasmid-based reverse genetic system for reovirus, another segmented dsRNA virus in the Reoviridae family (Kobayashi et al., 2007). In this system, 10 plasmids, each of which contains the cDNA of an authentic reovirus gene behind a T7 promoter and upstream of an HDV ribozyme, are transfected into murine cells (L929) infected with rDIs-T7pol. Viral transcripts made from the cDNAs launch the production of recombinant infectious virus, with yields reaching 10 to 100 PFU per transfected 60-mm well (10ex6 cells).[unreadable] [unreadable] Attempts have been made by us to adapt the reovirus plasmid-based reverse genetics system to rotavirus. We have (i) created 11 cDNA plasmids containing the rotavirus gene segments (SA11 strain) and confirmed the fidelity of the sequences, (ii) verified that the cDNAs were appropriately transcribed by T7 RNA polymerase and cleaved by the HDV ribozyme, (iii) defined appropriate transfection conditions for expression of viral proteins from these plasmids, and (iv) identified several cell types that are highly transfectable and permissive for rotavirus infection. Nonetheless, we have not yet been successful in rotavirus rescue. [unreadable] [unreadable] In addition to the advances for reovirus, the Roy laboratory used an alternative approach to create a reverse genetics system for another Reoviridae member, bluetongue virus (BTV) (Boyce et al., 2008). In this system, the 10 +RNAs of BTV are made in vitro from virion-derived cores and transfected directly into permissive cells. By co-transfecting cells with T7pol-generated transcripts, viruses can be recovered that contain a recombinant gene. One major limitation of this system is the need to laboriously screen for recombinant viruses by plaque purification. So far, attempts by us to develop an analogous system for rotavirus have been unsuccessful. [unreadable] [unreadable] (2) Characterization of the rotavirus antagonist (NSP1) of the IFN signaling pathway. An effective innate immune response requires the expression and secretion of Type 1 interferons (IFN-alpha and IFN-beta). The cascade of events required to stimulate IFN-gene expression is initiated by the activation of IRF3, a constitutively expressed transcription factor that induces IFN-beta production. In turn, IFN-beta induces the expression and activation of other transcription factors, including IRF7, the master regulator of IFN-alpha and IFN-beta production in the host. [unreadable] [unreadable] Rotavirus subverts IFN signaling by the action of its nonstructural protein NSP1 (Barro and Patton, 2005). Specifically, we have learned that NSP1 has the potential to interact with multiple members of the IRF family of proteins (Barro and Patton, 2007). This interaction is correlated with the degradation of IRF protein (e.g., IRF3, ORF5, and IRF7) in infected cells, via a proteasome-dependent process. This degradation prevents rotavirus-infected cells from expressing IFN-beta. In comparison, rotavirus strains producing C-truncated forms of NSP1 are defective in inducing the degradation of IRF proteins and in suppressing IFN expression. Rotaviruses producing defective NSP1 were also found to grow to lower titers in many cells lines than viruses producing intact NSP1 (Barro and Patton, 2007)[unreadable] [unreadable] The observation that NSP1 targets multiple members of the IRF family raises the possibility that the protein recognizes a shared element of these factors, such as their DNA-binding domain (DBD) or their IRF-interactive domain (IAD). To explore this possibility, we used transient expression assays to analyze the capacity of NSP1 to induce the degradation of IRF3-deletion mutants. The results indicate that the antagonist activity of NSP1 is mediated by its recognition of some aspect of the RD, a domain located at the C terminus of several IRFs that is required for IRF dimerization. [unreadable] [unreadable] (3) Analysis of the diversity and evolution of the rotavirus genome. The scarcity of complete genome sequence information for the rotaviruses prevents a comprehensive molecular analysis of rotavirus diversity and evolution and limits the usefulness of reverse genetics systems. To address the need for addition sequence information, we initiated projects over the last several years that have so far provided the complete genome sequences of nearly 100 rotavirus isolates. The subject viruses include prototypic laboratory strains used for studies on rotavirus biology, strains used in animal model systems and as vaccine candidates, human isolates developed as G-type reference strains, and isolates recovered from stool material of hospitalized rotavirus-infected children. [unreadable] [unreadable] In the past year, we completed sequencing of 10 lab-adapted, human group A rotavirus reference strains. Collectively, the strains represent 10 of the 11 serotypes (G-types) associated with human rotavirus disease: G1 (D strain), G2 (DS-1), G3 (P), G4 (ST3), G5 (IAL28), G6 (Se584), G8 (69M), G9 (WI61), G10 (A64), and G12 (L26). These and other existing sequences were then used to establish a classification system for rotaviruses, which have allowed, for the first time ever, each of the viral genes to be assigned to a particular genotype (Matthijnssens et al., 2008, JV 82:3204; Arch Virol 153:1621). Our sequence analysis of the 10 G-type reference strains indicates that most genes of human rotaviruses belong to one of three genotypes (1, 2, or 3). Of the reference strains representing rotavirus commonly infecting humans (G1-G4, G9), all the genes of each virus strain belong to the same genotype. Thus, none are inter-genogroup reassortants. The only inter-genogroup reassortants that were detected in the reference strains were those representing serotypes rarely associated with human infections (G6P9, G10P14, G12P4). These data suggest the existence of preferred constellations of rotavirus genes (Heiman et al., 2008, JV in press). [unreadable] [unreadable] To gain insight into the diversity and evolution of human rotaviruses circulating in a single location over a period of time, we developed a pilot robotics rotavirus sequencing program, via a contract with Dr. David Spiro at JC Venter, Inc., to sequence the complete genomes of 100 rotavirus samples. The rotavirus samples selected for analysis constituted part of a collection of archival stool samples collected from children during 1974 to 1991 at Childrens Hospital National Medical Center, Washington DC. During this period, G1P8 viruses typically dominated in any given year. A notable exception was in 1976, when the incidence of G3P8 infections in hospitalized children rose markedly, with a parallel decrease occurring in the incidence of G1P8 infections. Our sequence analysis indicates that the 1976 season was characterized by the appearance of two co-circulating lineages of G3P8 viruses, which contained minor but significant differences in their VP4 and VP7 neutralization epitopes. This study indicates that antigenically-distinct lineages of the same virus serotype (G3P8) can co-circulate and cause disease in the same season and in the same human population. Such co-circulation may significantly increase the incidence of severe disease.